The long term goal of this research is to understand the role of protein kinase C in rod outer segments of photoreceptors. Specifically, we will explore the phosphorylation of rhodopsin by protein kinase C. We will also characterize the structure and regulation of the rod outer segment protein kinase C. Protein kinase C is a ubiquitous signal transducer that processes the plethora of signals promoting lipid hydrolysis. The enzyme is known to desensitize a number of transduction pathways, generally by phosphorylating and deactivating unliganded and liganded receptors. Its relative abundance in rod outer segments and the identification of rhodopsin as its major substrate in situ lead us to test the hypothesis that protein kinase C provides a second messenger-regulated desensitization pathway in phototransduction, analogous to the second messenger-regulated, heterologous desensitization pathways in other G protein-coupled transduction pathways. The first two aims below address the function of protein kinase C in rod outer segments, and the third addresses the structure and regulation of rod outer segment protein kinase C. I. Phosphorylation of Rhodopsin by Protein Kinase C: conditions for and sites of phosphorylation. Biochemical and cell biological approaches are proposed to determine which conditions promote the protein kinase C- catalyzed phosphorylation of rhodopsin in the intact retina. Inhibitors and activators of protein kinase C will be used to dissect out the contribution of protein kinase C to the phosphorylation of rhodopsin in situ. We will also test the hypothesis that protein kinase C plays a role in heterologous desensitization by asking the question: do feedback pathways in the retina result in protein kinase C activation and rhodopsin phosphorylation? Lastly, we will identify the site(s) of phosphorylation of rhodopsin by protein kinase C and determine the sensitivity of this phosphorylated site to phosphatases. II. Phosphorylation of Rhodopsin by Protein Kinase C: functional consequences. The goal of this specific aim is to determine the consequences of the protein kinase C-catalyzed phosphorylation of rhodopsin. Specifically, we will address how this phosphorylation alters three macromolecular interactions: rhodopsin's interaction with rhodopsin kinase, arrestin, and transducin. We will test the hypothesis that the phosphorylation of rhodopsin by protein kinase C deactivates the receptor. III. Structure and Regulation of Protein Kinase C in Rod Outer Segments. This aim addresses the possibility that the protein kinase C in rod outer segments is a previously undescribed isozyme of the protein kinase C family. The goal of this aim is to prepare isozymically pure rod outer segment protein kinase C, to investigate its biochemical and molecular properties, and to determine its subcellular distribution.